Proc. Nati. Acad. Sci. USAVol. 83, pp. 7693-7697, October 1986Biochemistry

Amino acid sequence of the plasma membrane ATPase ofNeurospora crassa: Deduction from genomic and cDNA sequences

(Xgtll/nucleotide sequence/intron/membrane-spanning segment/amino acid sequence homologies)

KARL M. HAGER*, SUZANNE M. MANDALA*, JAMES W. DAVENPORT*, DAVID W. SPEICHERt,EDWARD J. BENZ, JR.*t, AND CAROLYN W. SLAYMAN*§Departments of *Human Genetics, tPathology, tInternal Medicine, and §Physiology, Yale University School of Medicine, 333 Cedar Street,New Haven, CT 06510

Communicated by Edward A. Adelberg, July 7, 1986

ABSTRACT The plasma membrane of Neurospora crassacontains an electrogenic H+-ATPase (EC 3.6.1.35), for whichwe have isolated and sequenced both genomic and cDNAclones. The ATPase gene is interrupted by four small introns(58-124 base pairs). It encodes a protein of 920 amino acids(Mr, 99,886) possessing as many as eight transmembranesegments. The Neurospora ATPase shows significant aminoacid sequence homology with the Na+,K+- and Ca2+-trans-porting ATPases of animal cells, particularly in regions thatappear to be involved in ATP binding and hydrolysis.

The plasma membrane ATPase (EC 3.6.1.35) of the fungusNeurospora crassa is a 100-kDa polypeptide that is deeplyembedded in the lipid bilayer and constitutes 7-10% of thetotal membrane protein (1). Its physiological function is topump protons out of the cell, generating an electrochemicalH+ gradient that in turn drives the H+-dependent uptake ofamino acids, sugars, and inorganic ions (2).The Neurospora ATPase has been intensively studied by

both physiological and biochemical techniques and as a resulthas become one of the best-known examples of anelectrogenic ion pump. Early studies with micropipetteelectrodes established that this pump generates a very largemembrane potential (to 350 mV, inside negative) and asomewhat smaller pH gradient (1.4 units, inside alkaline) fora total electrochemical gradient of =430 mV (2). Morerecently, current-voltage analysis has defined the stoichiom-etry of the pump (one H+ transported per ATP split) as wellas some of the kinetic parameters of the reaction cycle (3).These electrophysiological results have been complementedand extended by direct measurements of ATP-coupled pro-ton translocation, both in isolated plasma membrane vesiclesand in proteoliposomes constructed from purified 100-kDapolypeptide (4-6).

In its molecular structure, as well as in aspects of itsreaction mechanism, the Neurospora H+-ATPase (alongwith related enzymes from Saccharomyces cerevisiae andSchizosaccharomyces pombe) bears a striking resemblanceto the (Na+,K+)-, Ca2+-, and (H+,K+)-ATPases of animalcells (2). In each case, there is a 100-kDa catalytic subunitthat is phosphorylated by ATP to form a covalent 3-aspartylphosphate reaction intermediate. Furthermore, experimentswith group-specific reagents have revealed certain key aminoacid residues that appear to be conserved: an N-ethyl-maleimide-sensitive cysteine (7), a butanedione- andphenylglyoxal-sensitive arginine (8, 9), and a fluoresceinisothiocyanate (FITC)-sensitive lysine (refs. 10 and 11; J.Pardo, personal communication), all protected by ATP orADP. Thus, it has seemed reasonable to expect that the

portions of the 100-kDa polypeptide involved in ATP bindingand phosphorylation may be quite similar from one enzymeto the next, even though the portions that dictate ionspecificity and stoichiometry of ion transport must certainlydiffer.Gene cloning provides the most straightforward way to

determine the complete amino acid sequence of large proteinssuch as the ion-translocating ATPases and thus to learn thedegree to which they are structurally related. This paperdescribes the cloning and sequencing of the gene for theNeurospora H+-ATPase, from which the complete aminoacid sequence of its 100-kDa polypeptide has been deduced.Homologies between the Neurospora ATPase and othermembers of the ion-translocating ATPase group are dis-cussed.

METHODSPreparation of Antibody Specific for the Plasma Membrane

ATPase. Polyclonal rabbit antiserum was raised againstATPase purified by the method ofBowman et al. (1) followedby preparative NaDodSO4/polyacrylamide gel electrophore-sis. The antiserum was subjected to affinity chromatographyon a column of ATPase coupled to Sepharose CL-4B. Uponimmunoblotting of Neurospora plasma membranes, the af-finity-purified antibody specifically recognized a 100-kDaprotein that comigrated with the ATPase. In addition, itspecifically precipitated the 100-kDa band from Neurosporaplasma membranes labeled with "so'-.

Screening of cDNA and Genomic Libraries. The affinity-purified ATPase antibody was used to screen a NeurosporacDNA library (ref. 12; kindly provided by M. Sachs and U.RajBhandary) in the expression vector Xgtll (13). PhageDNA was prepared (14) from isolates that were positivethrough five rounds of screening, and cDNA inserts weresubcloned in the plasmid vector pSP65 (15). One clone,cDNA 18, was labeled by nick-translation (16) and used toidentify genomic clones within an EMBL4 (17) Neurosporalibrary (generously supplied by R. Geever and N. Giles) byplaque hybridization (18). Additional cDNA clones wereobtained by screening the Xgtll cDNA library with nick-translated genomic clone pKH14 (Fig. 1).DNA Sequencing. The DNA sequence of the genomic and

cDNA clones was determined by the dideoxynucleotide-chain termination method of Sanger et al. (19) modified forthe use of 35S-labeled deoxyadenosine 5'-[a-thio]triphos-phate (20). Templates were prepared by inserting restrictionfragments into phage M13mpl8 or mpl9 (21) or by thedeletion method of Dale et al. (22).

Tryptic Peptide Isolation and NH2-Terminal Sequencing.For peptide sequencing pure 100-kDa polypeptide was pre-

Abbreviations: bp, base pair(s); FITC, fluorescein isothiocyanate;kb, kilobase(s).

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A) Genomic ClonespKH14

pKH4 pKH5

CX C R SR K KP HX K BH C XIIII ~~~~~~~~~~~~~~~~~~~~~~~~~~~...................I...I

4 , fl 4 oi 1 4 1 -lo. 4 ON *4 * I* I --fo *4 '' -41 I

4 I 4- HI 4- F-*- H-*4-I h* I-*--*IIH **I *PI -* *IHa -p - *-* 4-H H 4- 4-I *41

-I 4- h-0* *. * 4 -- 4 4* --II*,--l *4.-4 , --- -H h.-*, * I- 4-H 4-H , I*,

i op - 4 , I ***f *0 *lN

B) cDNA Clones7 d 10 k

_-#-IN-4- ---I

18

4-H4-H-

S8+--00 *oP I4 * * I

4-4- -H-

4--

4 l 4

S18

4- 4-*4----4-H42 3 5

kh

FIG. 1. Structure of the Neurospora plasma membrane H+-ATPase gene. (A) Restriction map and sequencing strategy for genomic clonespKH4, pKH5, and pKH14. Only the six-base restriction sites Bgl II (B), Cla I (C), HindIII (H), Kpn I (K), Pst I (P), EcoRI (R), Sal I (S), XhoI (X) used in sequencing are shown. The extent and direction of sequencing are indicated by arrows. Exons are represented by shaded boxes;introns by open boxes. The direction of transcription is from left to right. (B) Structure of the cDNA clones; A represents the introns not presentin the cDNA clones. The DNA sequence of the cDNA clones is identical with that of the genomic clones except for a 43-bp sequence (not shown)on the 5' end of clone S18, which presumably is an artifact of cDNA synthesis.

pared by chromatography of glycerol-gradient-purified (1),NaDodSO4-solubilized ATPase on a Sephacryl S-400 col-umn, followed by extensive dialysis at 40C to remove

NaDodSO4. The intact 100-kDa polypeptide was digestedwith trypsin (ATPase/trypsin = 50/1, wt/wt) at 370C for 24hr, and the resulting peptides were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid, using acetonitrilegradients of 0-100% on a Supelco (Bellefonte, PA) LC-304column (4.6 mm x 25 cm). Amino acid analysis and auto-mated Edman degradation were performed as described (23).

RESULTS

Isolation of the Neurospora Plasma Membrane ATPaseGene. The first step in isolation of the ATPase gene was toprobe a Neurospora Xgtll cDNA library with antibodydirected against the purified 100-kDa protein. Of 106 plaquesscreened, 17 gave positive signals through five rounds ofplating. Twelve of the 17 plaques yielded small but detectableinserts upon EcoRI digestion of purified phage DNA, and theinserts fell into three nonoverlapping classes on the basis ofsize [193, 312, and 498 base pairs (bp)] and cross-hybridiza-tion data (Fig. 1). Representative clones (cDNA 7, 10, and 18)were chosen for further study. DNA from all three cloneshybridized to a species of Neurospora poly(A)+ RNA largeenough [4.3 kilobases (kb)] to encode a 100-kDa polypeptide.Furthermore, when RNA homologous to each of the DNAinserts was purified from a preparation of total Neurosporapoly(A)+ RNA by hybrid selection (24), in vitro translation ina rabbit reticulocyte lysate generated a 100-kDa polypeptidethat comigrated with authentic ATPase (data not shown).Thus, the three cDNA clones appeared to encode differentparts of the plasma membrane ATPase.

To obtain the entire ATPase gene, cDNA clone 18 (Fig. 1)was used to screen a Neurospora genomic library, yielding aseries of large (15- to 20-kb) overlapping genomic clones fromwhich smaller restriction fragments hybridizing with cDNA7, 10, and 18 were identified and subcloned. Finally, to isolatelarge cDNA clones, the Xgtll cDNA library was reprobedwith nick-translated genomic clone pKH14 (Fig. 1). Incontrast to the initial antibody screening of the library, whichgave cDNA clones with relatively small inserts, clones withinserts up to 1.8 kb were obtained. It is not clear whether thefailure to detect large inserts by antibody screening was dueto a lack of synthesis of some of the corresponding fusionproteins (for example, because of incorrect reading frames)or to poor detection of relatively large fusion proteins byimmunoscreening of plaques.

Nucleotide Sequence of the Plasmna Membrane ATPaseGene. Fig. 1 summarizes the strategy employed to sequenceboth the genomic and cDNA clones, and Fig. 2 illustrates thesequence of the ATPase gene and the amino acid sequencededuced from it. The most probable initiation codon beginsat base 1247; it is present in a sequence with homology to theconsensus eukaryotic translation start site CCRCCATGG(25) and is the only in-frame methionine codon upstream ofthe first intron. The ATPase coding region is divided by foursmall introns, ranging in size from 59 to 124 bp. All fourintrons contain three highly conserved elements, includingthe internal sequence RCTRAC that is probably involved inlariat formation during splicing (Fig. 3; ref. 26).The exons encode a protein of 920 amino acids with a

calculated molecular weight of 99,886, a value comparingfavorably with a molecular weight of 104,000 (1) determinedby NaDodSO4/polyacrylamide gel electrophoresis. Twolines of evidence support the correctness of the deducedamino acid sequence: (i) the predicted amino acid composi-

0

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Biochemistry: Hager et al. Proc. Nati. Acad. Sci. USA 83 (1986) 7695

1 GATCACCCTTTTTCGCGAGCGTCCGGTCGTCTCAGCGCACCTGTGTGATCCACGCAGGTCC 120

121 ATGTCATACTACTTCTCGGTGGACACTGTTGAATGACTCTGCGAACCTGTGCTACCTGAAG 240

241 TCTTTGGGCTCCGGAAAGGAGCAGAGAGGAGTGGTCGAAGATCACGCACACACGTCCGACG 360

361 GCGTGGATTCCCCGTCCCAGGTCCTTGAGGATTTGTCTGGATAGGGATGAGGCGATGGGG 480

481 CAGCCCCAGCCACCACAGCAAGTCGGAATCAAATCGGCTCGCATGTGACGCATCAATTGGG 600

601 CAGCAGAGTGTGACTGCGAAGCCCTGTGTACGTGAGTGATCTTTGCGTCTGTCCCTCCTTT 720

721 TCTAGTGAGTTTATCTCTCTGGACCCCCACCTAATTTCCACAACCCGCATCCTGTGAGTTA 840

841 CCTTCACATCCAAACCCACATGTACAAAACCACCGGGCGAACTCAGCGTACTTCGCGTCAG 960

961 TCAAAGCGACCGCTCACACGTCTCCATCACCCTCACCACGTTCCGGACGTCAGTTCACTGC 1080

1081 TACTCCCACCATAAACCATCTACTTCTCACATGCGCCGTACCAACATCACTGCTGTCAATC 1200

1201 CTATTCCTGAACTGTTCACAACCATGGACCCGCGGCCCTCCGCACAACAACGAGTGCAAGC 1320MetAlaAspHisSerAlaSerGlyAlaProAlaLeuSerThrAsnhleGluSerGlyL sPheAspGluLysAl

1321 1440~~~~~~~~~~~~~~~1

30 40 50 601441 GCCTCGTGACCCGAATACATATGAGGAGAAGGCATCGTGGCGGTTCCAGCTCCAATAACGG 1560

GluGluGluGluGlu~luAlaThrProGlyGlyGly~rgValValProGluAspMetLeuGflnThrAspThrArgV70 80

1561 TCGCCCTCAGGTGCACCGCCATCGCCACGTAGAGGAGGACCTCCATCTGTTTCTGTCATAT 1680

1681 1800910 1012

heValMetGlu

1801 CTGTGCAAGTCGCTCTCGTGCCAGCGGTATCGGCTTCGTGTCTTACCGTTGTTGTAGATCG 1920GlyAlaAlaValLeuAlaAlaGlyLeuGluAspTrpValAspPheGlyVal IleCysG1lyLeuLeuLeuLeuAsnAlaValValGlyPheValGlflGluPheGlfl1 3 0 1 4 0 150 160V

1922 CGTCACTGCATGAAGAGTCACACGCTCAGCATACAGGTACGTGTTCGATTGTTAGCGCTC 2040Ala~lySer IleValAso luLeuLysLy sThrLeuAlaLeuLysAl aVa1Va1Le

17r 1802041 CCTAGTCCCAGGTGGCCCAGCTCCGGTTCCAGTAGGGACTACCGTAGTGACTACAGTCTCT 2160

uArgAspGlyThrLeuLysGluIleGluAlaProGluValValProGly;~spIleLeuGlflValGluGluGlyThr IleIleProAlaAspGlyArg IleValThrAspAspAl aPheLe190 200 210 220

2161 CCGTGTATTCCTCGCATTTGTTGCACCAGTACGTTCCTCCGTTAGGGTAGTTGGTACCGCC 2280

230 240 250 260

2400~~~27287V3

2401 CCCCTACTTGTTCCGTTCGTCACCTGCAACTGGTACTGCTACTACGGCCGCGCTCGTTG AC 2520

310 ~~~~~~~~~~320330342521 CACCAGCGCGGCCTCTGCAAGAGCTGCAAGTTCCACATCTGTGGCAATTTCCGCAATGACT 2640

350 360 370 P 3802641 CACAACACCCCCAGCCTCCGCCGTTGCCGGACCTCTCGCGTGCGTCCCAAGAGTTGTCACA 2760

430 440 4510 460

2881 3000~~~440 5 4

470 F 480 490 5003001 TGGTGTCCTGTCGTTTGTTGCGAGGGCAGCCTGAATTGTTAGCTCTGCCCCGCCAACAAGC 3120

3121 3240~~~~15 50 4

3361 3480~~~~506 7 8

790 800 810 820 -3961 GACTGTCTGTCAGCGTAGTTACAGTCTGTAGAGCAATGATCGCAGTCTCAGTCCCCGTCA 34080

nG~nrgrGln~yLeu~e alAlaMet~hr~~lyAsp~lylesnesAlahryPh8ehr uLys~ys~ly~sp~hrGlyuleis ~ ll~yeSerAsp~hrIea Ala~Al~aArg Ie6rple~lah34081 42CTTCTGCCGTTGTCACTGTCCTAGCTTGCGTTCACCTTCCTAGTTTCGACCCGCACATGA300

u~lsPh eul eur lel lee snr ere snl luealalPVel a~ehAla~aleleulnahrgeu~al ler~rlanyrsplu~sn~laro710 M 740~~~~~~~~~~~~~90 2

43721 44GAACCGCATGACTCAGTTGGTTTCTCCTGTTGC-GCTGTCTGACCGCCACTTCCCGGGGA340

4561 4680~~~~776 708

4801 4920~~~~908080 24921 50GGCGTACGGTTTCTGTAACCTCACGTCCACGGTGTCACCCCAACCACTGCTGCGACGACT4085041 5160~~~~308OO 6

5281 GTGATTCGGTCTCTGCA ATCATGGGCGAGAGTCTAGCACATCGAACCATTTCAGGGAAGGCTTGATGCAACTAGCATCCGGAAGCCT5359ACGAGACCTGTGA 40

(Lysp9-Ls1e Tr3Ly5,Se0Ly6,an Gy8- alo value of reu28% rgov ertefrth95lamino acids to79%oveLys898)weresequencedand could be unambiguously located the remainder.92

within the deduced amino acid sequence. Not surprisingly, the Neurospora ATPase shows a lesserHomology with Other Ion-Transporting ATPases. The yeast degree of homology with the Na',K+- and Ca2+-ATPases of

Saccharomyces cerevisiae has a plasma membrane H+- animal cells-only about 14% overall. However, there areATPase with similar, though not identical, kinetic properties several regions of striking similarity, as illustrated in Fig. 4:(2). Comparison of the amino acid sequence of the Neuro- (i) a region of unknown function between residues 191 andspora plasma membrane ATPase with that of the correspond- 220; (ii) a region surrounding the phosphorylation site (resi-ing yeast enzyme of 918 amino acids (27) demonstrates an dues 327-386); and (iii) two other regions that, at least in the

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Proc. Natl. Acad. Sci. USA 83 (1986)

Intron1234

Size58 bp GTAAGT-35-----TACTAACC----7---TAG

124 bp GTACGT-----97-----TGCTGACT----10----CAG64 bp GTAGGT----- 36-----CGCTAACC----11----CAG67 bp GTAAGT-----38-----AGCTAACC----12----CAG

NeusrosporConsensus

YeastConsensus

GTAACGT-- -35- 291 --_ TGCTGACX- ---6- 20 --_CAGC AA A T

GTATGT------- ATAA-55 AT

FIG. 3. Introns of the Neurospora ATPase gene.

animal cell ATPases (10, 11, 32), appear to contribute to theATP binding site-one containing an FITC-reactive lysinethat can be protected by ATP (residues 473-503) and anothercontaining an 5 '-(p-fluorosulfonyl)benzoyladenosine-re-active lysine, which also can be protected by ATP (residues604-663). There is a smaller, though still significant, degreeof homology with the more distantly related K+-transportingATPase of Escherichia coli (31).

Predicted Transmembrane Orientation. Although the de-tailed structure of only a few membrane proteins is known,it has proven helpful to use sequential search procedures toidentify possible membrane-spanning segments on the basisof their hydrophobicity. In Fig. 5A, the procedure ofEngelman et al. (33) has been applied to the NeurosporaH+-ATPase. If one takes 20 kcal/mol (1 cal = 4.18J) as athreshold value for the free energy of transfer of a 20-aminoacid segment to water, above which value the segment islikely to be located in the membrane (33), the NeurosporaATPase is predicted to contain eight transmembrane seg-ments, 20 to 31 residues in length (Fig. 5B). Four of thesegments are found in the NH2-terminal one-third of theprotein and four in the COOH-terminal one-third. An addi-tional stretch of 19 amino acids from residue 657 to 675 hasa free energy of transfer that is only slightly smaller (18kcal/mol) and might be regarded as a candidate for a ninthtransmembrane segment. This would change the predictedlocation of the COOH terminus from the cytoplasmic side ofthe membrane to the extracellular side, a matter that will have

HCaNa, KK d p

HCaNa , K

HCaNa , KK d p

HCaNa , KK d p

HCaNa , KK d p

HCaNa , KK d p

1 9 11 4 017 81 21

2 4 52 0 82 4 1

to be resolved by direct biochemical experimentation. Theyeast, Na',K+- and Ca2+-ATPases have strikingly similarhydrophobicity profiles (data not shown) and have beenpostulated to contain six to ten transmembrane segments,also clustered in the NH2- and COOH-terminal regions (27,29, 30, 34, 35).

DISCUSSIONThis paper describes the cloning of the gene for the Neu-rospora plasma membrane H+-ATPase. The gene containsfour introns that are distributed nonrandomly, three clusteredat the 5' end and the fourth located quite close to the 3' end;the position of the introns has no obvious relationship to thedomain structure of the protein. Of interest, the correspond-ing yeast gene, though conspicuously homologous in itscoding regions, contains no introns (27). In a separate seriesof experiments not reported here, the Neurospora ATPasegene has been mapped by means of a restriction fragmentlength polymorphism technique to the left arm of chromo-some I, close to the mt locus (B. J. Bowman and R. L.Metzenberg, personal communication).From the sequencing of both genomic and cDNA clones,

the primary structure of the Neurospora H+-ATPase hasbeen deduced and a working model of its topology in themembrane has been developed. The model predicts thatapproximately 22% of the protein is embedded in the mem-brane, 74% protrudes into the cytoplasm, and only about 4%is exposed at the outer surface. The two major cytoplasmicloops show significant homology to the corresponding por-tions of the Na' ,K+- and Ca2+-ATPases, suggesting that theyparticipate in common functions such as ATP binding andhydrolysis. The availability of cloned genes for severalmembers ofthe ion-translocating ATPase family will pave theway for a direct test of this idea and may also permit theidentification of sequences involved in ion specificity.

We thank S. Baserga, L. Caballero, E. Feingold, F. Kalousck, J.Kraus, T. Lalor, R. Mercer, J. Metherall, J. Rose, and J. Schneiderfor technical advice and helpful discussions; M. Sachs, U.RajBhandary, R. Geever, and N. Giles for providing Neurospora

I E T PEVPGD-IL2V7EE1 IIPADGRIVTDD.AFE QVDQSALTGESLLAVwT 240IK A VPGDIVEVAIVGD KPADFIRILSIKSTT RVDQS LTGES V KH 190INAEEVVGDVEVKGGD IPAD RIISAN..GCKIVD ISJ|TGESIE TRS 226

VP S DL EIG G. ..AS Ytf. E SIA RE 168

VAF SjSAVKR G E A FVVITA TGDIN T FV ANAAAVNASGH 285LIFISGTNIIAAIGIKIALIG AT TGLSITIEI G D[IMADQMAJT K 7 248AFF|STN|CVEGTAR|GIV|VY RTVM TgASGLIEGGQ[_ 281

327 LJ ITIIG GLPAVVTTT M WAYLIK FV QKC S AG E CSDKTGTLT K 386300 EjAf -J3PEGLPAV59CLL TRFAKKNAIV L LIC CSDKTGTLTTNI359323 IGIII vPEG SKIR MSqSNrE!KSLE VETLGSTS ICSDKTGTLT Q 3822 5 6 L IGGLLSASAVM SRMIL G AdV A T R DWDIV LIL LDKUP SITL G 3 1 5

47 15 1 250 33 9 2

53 46 0 15 9 14 4 7

6 0 46 7 36 8 54 88

TC EFLFKTV EDHPIPEEDQAYKN EVF 5051F KGAP GVDRCNYVRVGTTIRVMTGPVIKKIL 546LVMKGAPER>IDRC|SSI L IfG KgLDEEL Fl 537HIRKGSDAWRRHVIANGG[FPTDVDQKVDQ@Rig 426

DPPRHDTYKTCEAKTLGLSIKLTGDAV R 566|DPPR|KEn1MGS IQLFCLRDAGIMTGG DNK TAII 633DP PRIAAMPPDIKIVIGKICRIS G IKVI TG PlTAIK 623fV-KGGIKEUFAQLK KMTGL A 479

G0FAEVIFHKYNV I QREJYLVAMTGVGNSLKKADGIAVEG.SLS A 663CCIFAVEIPKK[SVEY[JYDEjAMTGDGVNDAPALSKKAEGIAMG§A A 732EIVAT SP I GRQDAISKVAGDGVNDQPALKKADIGAAMGIAJKA 745DEFATAALIGTAEARAVAMTGKFTNDALJAO<@LAIA E AGN 5 4 7

FIG. 4. Amino acid homology between the Neurospora plasma membrane H+-ATPase and other ion-translocating ATPases. Homologieswere identified using the University of Wisconsin Genetics Computer Group programs (28), introducing gaps (indicated by *) to maximizehomology. Identical residues are boxed; the phosphorylation site is indicated by * and the FITC site by l. Designation of sequences is as follows:H, the Neurospora plasma membrane H+-ATPase sequenced in this work; Ca, the fast-twitch form of the rabbit sarcoplasmic reticulumCa2+-ATPase (29); Na,K, the sheep kidney Na+,K+-ATPase (30); and Kdp, the product of the E. coli KdpB gene (31). The H+-ATPase of yeastshows similar sequence homology with the animal cell and E. coli ion-translocating ATPases (27).

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44w

< 2(3:

0

LLI11U)

z< 21

0 -4

uj - 61zw

w

w-

B.80 160 240 320 400 480 560 640 720 800 880

FIRST AMINO ACID IN WINDOW

Out

In l)) 4

H2N

libraries; M. Hart and D. Engelman for conducting the hydropathyanalysis; and R. Serrano and G. Fink for making available a preprintof the yeast ATPase work. This study was supported by NationalInstitutes of Health Grant GM 15761. K.M.H. was the recipient of aNational Institutes of Health Predoctoral Traineeship (GM 07499);S.M.M., of a National Science Foundation Postdoctoral Fellowship(DMB 8508818); and J.W.D., of a National Institutes of HealthPostdoctoral Fellowship (GM 10363).

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COOH

FIG. 5. (A) Hydropathy plotof the Neurospora plasma mem-brane ATPase, generated by themethod of Engelman et al. (33)using a window of 20 amino ac-

ids. The free energy of transferfrom a lipid phase to water (inkcal/mol of 20-amino acid seg-ment) is plotted as a function ofthe first amino acid in the win-dow. Hydrophobic regions are

above the x-axis and hydrophilicregions below it. (B) Model ofthe transmembrane orientationof the Neurospora plasma mem-brane ATPase. In and Out referto the cytoplasmic and externalsides of the membrane, re-

spectively.

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31-40.23. Speicher, D. W., Davis, G., Yurchenco, P. D. & Marchesi, V. T. (1983)

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